MONOCLONAL ANTIBODY M195. Mouse monoclonal antibody M195 is an IgG2a (1, 2) which reacts with 60-70% of samples of leukemia cells from patients with acute myelogenous leukemia (AML). M195 also binds to early myeloid cells (CFU-GM) and some monocytes but not to the earliest myeloid progenitors. The target antigen is not expressed on any other hematopoietic or non-hematopoietic tissue. Antibodies to a related antigen on the same protein (CD33), My9 and L4F3, are currently being used to purge bone marrow of ANLL before autologous transfusion (3, 4). M195 is rapidly internalized into cells after binding and this effect can enhance delivery of radiometals, radioiodine or conjugated toxins into cells (5). M195 is able to kill leukemia cells with rabbit or guinea pig complement, but not by use of human complement or human antibody-dependent cellular cytotoxicity in vitro. Activation of these mediators in vitro has correlated with these effects in vivo (6), but it is not known if the lack of in vitro effects will predict lack of in vivo effects. Because M195 also reacts with early myeloid cells, normal marrow progenitors may be affected also.
MYELOID LEUKEMIAS. Long-term survival for patients with acute leukemia given the best current chemotherapeutic regimens is generally less than 20% (7). Survival of patients who relapse or who fail first attempts at induction chemotherapy is far lower. Autologous or allogeneic bone marrow transplantation may improve survival, but only in a small subset of patients (8). There are no effective therapies for myelodysplastic syndromes or chronic monocytic leukemias and long term survival in these diseases is rare. Among patients with chronic myeloid leukemias (CML), only allogeneic bone marrow transplant has had an impact on survival (9). An approach to therapy which may be more effective is the use of monoclonal antibodies.
MONOCLONAL ANTIBODIES TO MYELOID ANTIGENS. Monoclonal antibodies (mAb) reactive with differentiation antigens present on myeloid cells and their progenitors are being used to study hematopoietic differentiation, to identify acute nonlymphoid leukemia (ANLL), to study the effects of hematopoietic growth factors, to purge bone marrow of leukemia cells, and for therapy in vivo (10-24).
The antigens displayed on the surface of acute nonlymphocytic leukemia (ANLL) cells and hematopoietic progenitor cells are being mapped in a number of laboratories using monoclonal antibodies (mAbs)(25).
These studies have been directed at identifying antigens that are useful in distinguishing lymphoid from nonlymphoid leukemias (26-28), in subtyping of acute myelogenous leukemia, and in predicting outcome (29-34) and in therapy in vivo (35) or via bone marrow purging ex vivo (36). Antigens defining ANLL cells also identify normal hematopoietic cells during early stages of their development and thus should be classified as differentiation antigens rather than leukemia specific antigens.
Antigens restricted to the earliest stages of hematopoietic development are of particular interest since ANLL is thought to be derived from these cells (37-40). Monoclonal antibodies identifying these early cells can help in their purification or the study of growth regulation and control of differentiation (41). Such early progenitors may be useful for autologous reinfusion in bone marrow rescue (42). Studies of bone marrow from patients with ANLL have shown that the clonogenic cells are probably derived from a subset of cells which are phenotypically more immature that the majority of cells in circulation (38, 39). This suggests that analysis of the development of leukemia cells, as well as therapeutic trials, should also be directed at these early cells and not simply the phenotypically predominant cells in the marrow and peripheral blood.
Several mAbs restricted to hematopoietic progenitors have been described: MY10, 3C5, and 12.8 recognize a 115-kDa glycoprotein (gp115 [CD34]) found on normal colony forming cells, myeloblasts, and leukemic blasts from most patients with ANLL and acute lymphoid leukemias (42-44). Monoclonal anitbody NHL-30.5 identifies a 180-kDa protein found on a similar distribution of cells (45-46). The My9 and L4F3 antibodies identify a 67-kDa glycoprotein (CD33) (48-50) which is expressed on slightly more mature progenitors (subsets of CFU-GEMM and some older cells) and is restricted to leukemias of the myeloid and monocytic lineage. Long-term culture studies suggest that elimination of cells bearing the CD33 antigen will still allow regrowth of normal marrow cells of all lineages, presumably because of the presence of more immature antigen negative progenitors (48). Sabbath et al. (39) show that the CD33 antigen is expressed on leukemic colony-forming cells whereas other more mature markers are less commonly expressed. Finally, studies with ANLL marrow suggest it may be possible to purge leukemia cells from the bone marrow of many patients with ANLL using complement fixing antibodies to CD33 without destroying the ultimate normal progenitors (47). Several other antibodies with a less restricted distribution have also been described (38, 51, 52).
RADIOLABLED ANTIBODIES. Since the discovery of hybridoma technology by Kohler and Milstein (53), there has been considerable interest in the utility of monoclonal antibodies as carriers of radioactivity for the diagnosis and therapy of cancer (54, 55). After the initial report by Goldenberg et al. on the utility of radiolabeled antibodies in the detection of cancer (56), there have been several clinical trials utilizing radiolabeled monoclonal antibodies in lymphoma and leukemia (57-63), both for radioimmunolocalization and radioimmunotherapy. Most of these trials have employed radioiodine (57-62); Carrasquillo and associates have also studied .sup.111 In-labeled monoclonal antibody T101 (57, 61) in the diagnosis of T-cell lymphoma. One obvious advantage of radiolabeled antibodies is that the specificity of antibody for the target antigen, often expressed in increased quantities on neoplastic cells, offers a potentially useful method for the selective delivery of radioactivity to the tumor site; moreover, the range of potentially lethal radiation emitted by most currently used radionuclides extends over several cell diameters, making it theoretically possible for the radiation to be cytotoxic to neighboring neoplastic cells that lack the target antigen.
Historically, beta-minus particle emitters such as .sup.131 I have been preferred for mAb directed radioimmunotherapy. Radionuclides such as .sup.125 I that decay by electron capture are also of interest in radioimmunotherapy because they are cytotoxic when internalized by the cell (64). .sup.125 I labeled antibodies that are internalized into the cell following interaction with the target antigen may thus be cytotoxic (65). Studies in both animals and humans have shown that the radiometal .sup.111 In concentrates to a significantly greater extent in tumor compared to radioiodine (66, 69). Thus, use of beta-minus emitting radiometals such as .sup.90 Y are of interest for therapy as well. Therefore, the choice of radionuclide used to label monoclonal antibodies may be of importance in the design of clinical trials utilizing radiolabeled mAbs for diagnosis and therapy.
INTERNALIZATION OF MONOCLONAL ANTIBODIES. Antigen-antibody complexes may either be shed from the cell or internalized into the cell following interaction with antibody. This process, known as modulation, was first described in mice (70) and later confirmed to occur during trials of mAb in humans (71). The process appears to be a general phenomenon found in many antigen-antibody systems of hematopoietic cells (72) and neoplasms as well as in solid tumors (73). Modulation may result in mAb shedding, internalization, or both processes. Shedding may result in residence time of the antibody on the target cell too short to achieve cell kill. On the other hand, internalized antigen-antibody complexes may theoretically deliver significant amounts of cytotoxic antibody into the cell if the cytotoxic label attached to the antibody is internalized into the cell and retained.
The cell biology of modulation and receptor internalization has been studied elsewhere (74, 75).
MONOCLONAL ANTIBODY THERAPY OF LEUKEMIA. Monoclonal antibody based therapies are ideally applicable to the hematopoietic neoplasms because of readily accessible neoplastic cells in the blood, marrow, spleen and lymph nodes which allow rapid and efficient targeting of specific mAb. The well characterized immunophenotypes of the various lineages and stages of hematopoietic differentiation should enable identification of antigen targets for selective binding of mAb to neoplastic cells while sparing other necessary hematopoietic lineages and progenitor cells.
In some models of leukemia, specific uptake of antibodies onto target cells can be demonstrated within minutes, followed by losses of mAb from the cells by modulation (76-79). Similar modulation has been seen in pilot studies in acute leukemia in humans (80, 81). Based on this biology and pharmacokinetics, it has been proposed that mAb tagged with short-lived nuclides emitting short-ranged, high linear energy transfer (LET) alpha particles (79) or short-ranged auger electrons (82, 83) may be effective in therapy.
A challenge in treating AML with mAb is the necessity for selection of an antigen target found on clonogenic myeloid leukemia blasts, but not on the ultimate normal hematopoietic progenitors. Gp67 (CD33) appears to be one such target. Cytotoxic murine mAb to this target can selectively kill early myeloid cells (CFU-GM, BFU-E) and leukemia cells without eliminating the potential for regrowth of normal bone marrow progenitors. This selectivity has been applied to the purging of bone marrow before autologous reinfusion in the treatment of AML (84-91). M195 is a mouse IgG2a reactive with gp67 which is capable of rapidly internalizing into target cells upon binding, but is unable to kill cells in vitro by use of human complement or effector cells (88, 89). Other than committed hematopoietic progenitor cells and some monocytes, M195 does not appear to react with any normal cells or tissues (26, 27).
Previously all of the antibody therapy work done had used the mouse monoclonal antibody. This approach has certain limitations. The mouse antibody has limited biological activity for killing cells, and it has a relatively short half life in human serum. Additionally because the antibody used is derived from a mouse it can trigger an immune response to the foreign material. Thus use of a humanized antibody helps to improve the therapeutic attributes of the molecule. It improves half life of the molecule in human serum and reduces the immune response.
GENETIC THERAPY. A new powerful tool to introduce genes into mammalian cells is the construction of recombinant retroviral vectors in which exogenous genes replace portions of the viral genome (95). However, some constraints may limit the use of retroviral vectors. One concerns cell tropism: this limitation has been in part circumvented by the development of amphotropic viral vectors, able to infect a wide range of cells from different species. On the contrary, it may be advantageous for particular purposes, including gene therapy, to infect only a chosen suppopulation of cells. This is not possible with either ecotropic or amphotropic viruses if the whole cell population bears membrane receptors for the virus. One method to allow selective targeting of specific cells within a mixture is the use of specific antigenic targets. The development of a technology that would allow us to introduce a recombinant retrovirus into selected cells is a way to considerably extend the potentiality of retroviral vectors.